Pectin Homogalacturonan Research Articles

Ancient origin of acetyltransferases catalyzing O-acetylation of plant cell wall polysaccharides.

Members of the domain of unknown function 231 (DUF231)/trichome birefringence-like (TBL) family have been shown to be O-acetyltransferases catalyzing the acetylation of plant cell wall polysaccharides, including pectins, mannan, xyloglucan and xylan. However, little is known about the origin and evolution of plant cell wall polysaccharide acetyltransferases. Here, we investigated the biochemical functions of TBL homologs from Klebsormidium nitens, a representative of an early divergent class of charophyte green algae that are considered to be the closest living relatives of land plants, and Marchantia polymorpha, a liverwort that is an extant representative of an ancient lineage of land plants. The genomes of K. nitens and M. polymorpha harbor two and six TBL homologs, respectively. Biochemical characterization of their recombinant proteins expressed in human embryonic kidney (HEK) 293 cells demonstrated that the two K. nitens TBLs exhibited acetyltransferase activities acetylating the pectin homogalacturonan (HG) and hence were named KnPOAT1 and KnPOAT2. Among the six M. polymorpha TBLs, five of them (MpPOAT1 to 5) possessed acetyltransferase activities toward pectins and the remaining one (MpMOAT1) catalyzed 2-O- and 3-O-acetylation of mannan. While MpPOAT1,2 specifically acetylated HG, MpPOAT3,4,5 could acetylate both HG and rhamnogalacturonan-I (RG-I). Consistent with the acetyltransferase activities of these TBLs, pectins isolated from K. nitens and both pectins and mannan from M. polymorpha were shown to be acetylated. These findings indicate that the TBL genes were recruited as cell wall polysaccharide O-acetyltransferases as early as in charophyte green algae with activities toward pectins and they underwent expansion and functional diversification to acetylate various cell wall polysaccharides during evolution of land plants.

Homogalacturonan Pectins Tuned as an Effect of Susceptible rbohD, Col-0-Reactions, and Resistance rbohF-, rbohD/F-Reactions to TuMV.

The plant cell wall is an actively reorganized network during plant growth and triggered immunity in response to biotic stress. While the molecular mechanisms managing perception, recognition, and signal transduction in response to pathogens are well studied in the context of damaging intruders, the current understanding of plant cell wall rebuilding and active defense strategies in response to plant virus infections remains poorly characterized. Pectins can act as major elements of the primary cell wall and are dynamic compounds in response to pathogens. Homogalacturonans (HGs), a main component of pectins, have been postulated as defensive molecules in plant-pathogen interactions and linked to resistance responses. This research focused on examining the regulation of selected pectin metabolism components in susceptible (rbohD-, Col-0-TuMV) and resistance (rbohF-, rbohD/F-TuMV) reactions. Regardless of the interaction type, ultrastructural results indicated dynamic cell wall rebuilding. In the susceptible reaction promoted by RbohF, there was upregulation of AtPME3 (pectin methylesterase) but not AtPME17, confirmed by induction of PME3 protein deposition. Moreover, the highest PME activity along with a decrease in cell wall methylesters compared to resistance interactions in rbohD-TuMV were noticed. Consequently, the susceptible reaction of rbohD and Col-0 to TuMV was characterized by a significant domination of low/non-methylesterificated HGs. In contrast, cell wall changes during the resistance response of rbohF and rbohD/F to TuMV were associated with dynamic induction of AtPMEI2, AtPMEI3, AtGAUT1, and AtGAUT7 genes, confirmed by significant induction of PMEI2, PMEI3, and GAUT1 protein deposition. In both resistance reactions, a dynamic decrease in PME activity was documented, which was most intense in rbohD/F-TuMV. This decrease was accompanied by an increase in cell wall methylesters, indicating that the domination of highly methylesterificated HGs was associated with cell wall rebuilding in rbohF and rbohD/F defense responses to TuMV. These findings suggest that selected PME with PMEI enzymes have a diverse impact on the demethylesterification of HGs and metabolism as a result of rboh-TuMV interactions, and are important factors in regulating cell wall changes depending on the type of interaction, especially in resistance responses. Therefore, PMEI2 and PMEI3 could potentially be important signaling resistance factors in the rboh-TuMV pathosystem.

Physicochemical and antioxidant properties of pectin fractions extracted from lemon (Citrus Eureka) peels

Pectin, a natural polysaccharide, holds versatile applications in food and pharmaceuticals. However, there is a need for further exploration into extracting novel functional fractions and characterizing them thoroughly. In this study, a sequential extraction approach was used to obtain three distinct lemon pectin (LP) fractions from lemon peels (Citrus Eureka): LP extracted with sodium acetate (LP-SA), LP extracted with ethylenediaminetetraacetic acid (LP-EDTA), and LP extracted with sodium carbonate and sodium borohydride (LP-SS). Comprehensive analysis revealed low methyl-esterification in all fractions. LP-SA and LP-SS displayed characteristics of rhamnogalacturonan-I type pectin, while LP-EDTA mainly consisted of homogalacturonan pectin. Notably, LP-SA formed self-aggregated particles with rough surfaces, LP-EDTA showed interlocking linear structures with smooth planes, and LP-SS exhibited branch chain structures with smooth surfaces. Bioactivity analysis indicated that LP-SA had significant apparent viscosity and ABTS radical scavenging activity, while both LP-EDTA and LP-SS showed excellent thermal stability according to thermogravimetric analysis (TGA). Furthermore, LP-SS exhibited remarkable gel-forming ability and significant hydroxyl free radicals scavenging activity. In conclusion, this study presents a novel method for extracting various lemon pectin fractions with unique structural and bioactive properties, contributing insights for advanced applications in the food and pharmaceutical sectors.

TBL38 atypical homogalacturonan-acetylesterase activity and cell wall microdomain localization in Arabidopsis seed mucilage secretory cells

Plant cell walls constitute complex polysaccharidic/proteinaceous networks whose biosynthesis and dynamics implicate several cell compartments. The synthesis and remodeling of homogalacturonan pectins involve Golgi-localized methylation/acetylation and subsequent cell wall-localized demethylation/deacetylation. So far, TRICHOME BIREFRINGENCE-LIKE (TBL) family members have been described as Golgi-localized acetyltransferases targeting diverse hemicelluloses or pectins. Using seed mucilage secretory cells (MSCs) from Arabidopsis thaliana, we demonstrate the atypical localization of TBL38 restricted to a cell wall microdomain. A tbl38 mutant displays an intriguing homogalacturonan immunological phenotype in this cell wall microdomain and in an MSC surface-enriched abrasion powder. Mass spectrometry oligosaccharide profiling of this fraction reveals an increased homogalacturonan acetylation phenotype. Finally, TBL38 displays pectin acetylesterase activity invitro. These results indicate that TBL38 is an atypical cell wall-localized TBL that displays a homogalacturonan acetylesterase activity rather than a Golgi-localized acetyltransferase activity as observed in previously studied TBLs. TBL38 function during seed development is discussed.

Functional dissection of the PME and PMEI super families in the protection of Populus pectin during Fusarium wilt

Integration of the pectin homogalacturonan (HG) integrity and remodeling into defense immunity facilitates building blocks of stress resilience within the host and pathogen interactions in the forests. Recent advances in apoplastic pectin methylesterases (PMEs) and coordinated PME inhibitors (PMEIs) deployed immune signaling, highlighting their critical roles in regulating biotic stress adaptation. Nevertheless, the PME and PMEI multiple isoenzyme families involved in defense priming in the model tree species are largely unknown. The in silico demonstration of the molecular profile identified 70 PME- (50 type-I proPME and 20 type-II PME) and 49 PMEI-encoding genes in the Populus genome. Evaluation of the transcript abundance verified 20 PtproPMEs and 18 PtPMEIs that showed predominant expression in the root and genetic redundancy for type-II PtPMEs. Dynamic changes in the transcriptome and experimental evaluation revealed a minuscule number of type-I proPMEs and PMEIs that showed marked promotions during a time-course Fusarium solani (Fs) infection. Using GC/LC-mass-based non-targeted metabolomics to inspect structure-related carbohydrates and PME activities suggested a correlation between proPME-PMEI co-expression and deformed pectin HG biosynthesis. Further functional examination of the enzyme inhibitory affinities of recombinant PtPMEI1 and 39 in vitro and apoplastic patterns suggested a clustering of the type-I proPME in the regulation of the innate immunity by maintaining homeostasis of the pectin HG de-methylesterification (DME) in the cell wall. The coherent omics-wide survey of the fungi pathogen-induced PME-PMEIs and disturbed carbohydrate accumulation provide theoretical cues for in-depth mining of the biological significance of the underlying immune signaling networks within an apoplastic niche in trees.

GoSAMTs are required for pectin methyl-esterification and mucilage release in seed coat epidermal cells.

GoSAMTs play a role in the methylation of polysaccharides synthesized by the Golgi. Pectin homogalacturonan (HG) methyl-esterification is essential for the proper function of this polysaccharide in cell walls. In order to better understand the role of GoSAMTs in HG biosynthesis, we analyzed mucilage methyl-esterification in gosamt mutants. To determine the function of GoSAMT1 and GoSAMT2 in HG methyl-esterification we utilized epidermal cells of seed coats, as these structures produce mucilage, which is a pectic matrix. We evaluated differences in seed surface morphology and quantified mucilage release. We measured methanol release, and used antibodies and confocal microscopy to analyze HG methyl-esterification in mucilage. We observed morphological differences on the seed surface and delayed, uneven mucilage release in gosamt1-1gosamt2-1 double mutants. We also found changes in the distal wall length indicating abnormal cell wall breakage in this double mutant. Using methanol release and immunolabeling, we confirmed that GoSAMT1 and GoSAMT2 are involved in HG methyl-esterification in mucilage. However, we did not find evidence of decreasing HG in the gosamt mutants. Confocal microscopy analyses detected different patterns in the adherent mucilage and a greater number of low-methyl-esterified domains near the seed coat surface, which correlates with a greater number of "egg-box" structures in this region. We also detected a shift in the partitioning between the Rhamnogalacturonan-I soluble and adherent layers of the double mutant, which correlated with increased amounts of arabinose and arabinogalactan-protein in the adherent mucilage. The results show that the HG synthesized in gosamt mutant plants is less methyl esterified, resulting in more egg-box structures, which stiffen the cell walls in epidermal cells and change the rheological properties of the seed surface. The increased amounts of arabinose and arabinogalactan-protein in adherent mucilage, also suggests that compensation mechanisms were triggered in the gosamt mutants.

A Homogalacturonan from Peel of Winter Jujube (Zizyphus jujuba Mill. cv. Dongzao): Characterization and Protective Effects against CCl4-Induced Liver Injury.

A homogalacturonan pectin (HG, designated as WJP-F80) was extracted from the peel of winter jujube (Zizyphus jujuba Mill. Cv. Dongzao) and separated via ethanol-graded precipitation. The structural and conformational features were elucidated through HPAEC-PAD, GC-MS, 2D NMR, and HPSEC-MALLS studies. In vivo assessments were carried out to evaluate the hepatoprotective effects of WJP-F80 against CCl4-induced injury of mice. Results showed that WJP-F80 was a linear 1,4-α-galacturonan with partially methyl-esterified at O-6 of GalpA and occasionally acetylation. The Mw of WJP-F80 was determined as 45.3 kDa, the polydispersity was calculated as 1.56, and the Rg was measured as 22.7 nm in 0.1 M NaNO3. The conformational analysis revealed that WJP-F80 exhibited as rigid stiff chain in low Mw range, while aggregation by self-assembly of HG chains lead to high Mw and random coil conformation. In vivo studies indicated that WJP-F80 can protect the livers of mice from acute injury induced via CCl4 by decreasing the serum biochemical markers of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) to normal levels. This work provides a theoretical basis for the value-added deep processing of winter jujube.

A novel multiplex lateral flow assay for rapid assessment of pectin structural/functional properties

Pectin is a multi-faceted functional food ingredient widely used in the food, pharmaceutical and personal care industries for different purposes, based on its functional properties, which are primarily determined by the structural properties (degree and pattern of methyl-esterification) of its homogalacturonan (HG) backbone. Currently, pectin's commercial functionality is defined by lengthy, laborious and operator dependent tests. Using pectin homogalacturonan (HG) recognizing antibodies, we have developed a novel, rapid, easy to use lateral flow assay (LFA) to detect and assess pectin structural properties that are related to its functionality. The LFA system includes three different capture antibodies immobilized on a test strip and two detection antibodies labeled with different colors of Gold NanoParticles in a small vial. A user needs only to drop a pectin sample into the small vial and insert the test strip into the vial, then wait 5 min to obtain results. With a detection limit of 0.02 μg/mL of pectin, the developed LFA is able to discern pectin's degree and pattern of methyl-esterification. The capability of the LFA has been validated by testing a series of known experimental and commercial pectins. The technology can be easily scaled up to high-volume manufacturing. The convenience of such an LFA would benefit the entire spectrum of the pectin utilization chain, from producers and ingredient suppliers to food formulators and those in the medical and personal care industries.

Polymerization of the backbone of the pectic polysaccharide rhamnogalacturonan I.

Rhamnogalacturonan I (RG-I) is a major plant cell wall pectic polysaccharide defined by its repeating disaccharide backbone structure of [4)-α-D-GalA-(1,2)-α-L-Rha-(1,]. A family of RG-I:Rhamnosyltransferases (RRT) has previously been identified, but synthesis of the RG-I backbone has not been demonstrated in vitro because the identity of Rhamnogalacturonan I:Galaturonosyltransferase (RG-I:GalAT) was unknown. Here a putative glycosyltransferase, At1g28240/MUCI70, is shown to be an RG-I:GalAT. The name RGGAT1 is proposed to reflect the catalytic activity of this enzyme. When incubated together with the rhamnosyltransferase RRT4, the combined activities of RGGAT1 and RRT4 result in elongation of RG-I acceptors in vitro into a polymeric product. RGGAT1 is a member of a new GT family categorized as GT116, which does not group into existing GT-A clades and is phylogenetically distinct from the GALACTURONOSYLTRANSFERASE (GAUT) family of GalA transferases that synthesize the backbone of the pectin homogalacturonan. RGGAT1 has a predicted GT-A fold structure but employs a metal-independent catalytic mechanism that is rare among glycosyltransferases with this fold type. The identification of RGGAT1 and the 8-member Arabidopsis GT116 family provides a new avenue for studying the mechanism of RG-I synthesis and the function of RG-I in plants.

The calcium-mediated homogalacturonan pectin complexation in cell walls contributes the firmness increase in loquat fruit during postharvest storage

IntroductionPostharvest textural changes in fruit are mainly divided into softening and lignification. Loquat fruit could have severe lignification with increased firmness during postharvest storage. Pectin is mainly associated with the postharvest softening of fruit, but some studies also found that pectin could be involved in strengthening the mechanical properties of the plant. ObjectivesThis study focused on characterizing the dynamics of pectin and its complexation in the cell wall of lignified loquat fruit during postharvest storage, and how these changes could influence fruit firmness. MethodsThe homogalacturonan (HG) pectin in the cell wall of loquat fruit was identified using monoclonal antibodies. An oligogalacturonide (OG) probe was used to label the egg-box structure formed by Ca2+ cross-linking with low-methylesterified HG. An exogenous injection was used to verify the role of egg-box structures in the firmness increase in loquat fruit. ResultsThe JIM5 antibody revealed that low-methylesterified HG accumulated in the tricellular junctions and middle lamella of loquat fruit that had severe lignification symptoms. The pectin methylesterase (PME) activity increased during the early stages of storage at 0 °C, and the calcium-pectate content and flesh firmness constantly increased during storage. The OG probe demonstrated the accumulation of egg-box structures at the cellular level. The exogenous injection of PME and Ca2+ into the loquat flesh led to an increase in firmness with more low-methylesterified HG and egg-box structure signals. ConclusionPME-mediated demethylesterification generated large amounts of low-methylesterified HG in the cell wall. This low-methylesterified HG further cross-linked with Ca2+ to form egg-box structures. The pectin-involved complexations then contributed to the increased firmness in loquat fruit. Overall, besides being involved in fruit softening, pectin could also be involved in strengthening the mechanical properties of postharvest fruit. This study provides new ideas for obtaining a better texture of postharvest loquat fruits based on pectin regulation.

Root cell wall remodeling mediates copper oxide nanoparticles phytotoxicity on lettuce (Lactuca sativa L.)

Root cell wall (RCW) remodeling induced by copper oxide nanoparticles (CuO NPs) and its consequences for root growth of lettuce (Lactuca sativa L.) were investigated in this study. The results showed that a low concentration of CuO NPs (5 mg L−1) stimulated reactive oxygen species (ROS) signaling, led to the degradation of pectin by H2O2, and promoted root elongation by 22.1% by triggering RCW loosening. However, under treatment with a high concentration of CuO NPs (450 mg L−1), the unordered distribution of pectin homogalacturonan enhanced cell wall adhesion. Also, the increased xyloglucan and the decreased xyloglucan endotransglycosylase/hydrolase (XTH) activities caused cell wall stiffening, making it difficult for lettuce roots to extend. Meanwhile, the contents of hemicellulose and low-methylated pectin were increased remarkably due to the downregulation of the encoding genes XTH15, XTH17, XTH31 and the upregulation of PME3, which provided abundant binding sites of RCW with CuO NPs. In addition, the accelerated development of the apoplastic barrier and root lignification blocked the absorption of CuO NPs, and the endodermal cell walls were doubled in thickness, greatly enhancing their retention capacity to CuO NPs. Taken together, the above findings suggest that CuO NPs have concentration-dependent effects on lettuce roots, which are associated with plant tolerance and mediated by RCW remodeling.

Determining Methyl-Esterification Patterns in Plant-Derived Homogalacturonan Pectins.

Homogalacturonan (HG)-type pectins are nutrient components in plants and are widely used in the food industry. The methyl-esterification pattern is a crucial structural parameter used to assess HG pectins in terms of their nutraceutical activity. To better understand the methyl-esterification pattern of natural HG pectins from different plants, we purified twenty HG pectin-rich fractions from twelve plants and classified them by their monosaccharide composition, Fourier transform-infrared spectroscopy (FT-IR) signatures, and NMR analysis. FT-IR shows that these HG pectins are all minimally esterified, with the degree of methyl-esterification (DM) being 5 to 40%. To examine their methyl-esterification pattern by enzymatic fingerprinting, we hydrolyzed the HG pectins using endo-polygalacturonase. Hydrolyzed oligomers were derivatized with 2-aminobenzamide and subjected to liquid chromatography-fluorescence-tandem mass spectrometry (HILIC-FLR-MSn). Twenty-one types of mono-/oligo-galacturonides having DP values of 1–10 were found to contain nonesterified monomers, dimers, and trimers, as well as oligomers with 1 to 6 methyl-ester groups. In these oligo-galacturonides, MSn analysis demonstrated that the number of methyl-ester groups in the continuous sequence was 2 to 5. Mono- and di-esterified oligomers had higher percentages in total methyl-esterified groups, suggesting that these are a random methyl-esterification pattern in these HG pectins. Our study analyzes the characteristics of the methyl-esterification pattern in naturally occurring plant-derived HG pectins and findings that will be useful for further studying HG structure-function relationships.

Cryo-electron tomography of the onion cell wall shows bimodally oriented cellulose fibers and reticulated homogalacturonan networks

SummaryOne hallmark of plant cells is their cell wall. They protect cells against the environment and high turgor and mediate morphogenesis through the dynamics of their mechanical and chemical properties. The walls are a complex polysaccharidic structure. Although their biochemical composition is well known, how the different components organize in the volume of the cell wall and interact with each other is not well understood and yet is key to the wall’s mechanical properties. To investigate the ultrastructure of the plant cell wall, we imaged the walls of onion (Allium cepa) bulbs in a near-native state via cryo-focused ion beam milling (cryo-FIB milling) and cryo-electron tomography (cryo-ET). This allowed the high-resolution visualization of cellulose fibers in situ. We reveal the coexistence of dense fiber fields bathed in a reticulated matrix we termed “meshing,” which is more abundant at the inner surface of the cell wall. The fibers adopted a regular bimodal angular distribution at all depths in the cell wall and bundled according to their orientation, creating layers within the cell wall. Concomitantly, employing homogalacturonan (HG)-specific enzymatic digestion, we observed changes in the meshing, suggesting that it is—at least in part—composed of HG pectins. We propose the following model for the construction of the abaxial epidermal primary cell wall: the cell deposits successive layers of cellulose fibers at −45° and +45° relative to the cell’s long axis and secretes the surrounding HG-rich meshing proximal to the plasma membrane, which then migrates to more distal regions of the cell wall.

Pilot-scale biorefinery for the production of purified biopolymers based on hydrothermal treatment in flow-through reactor cycles

Purified solid fractions of hemicellulose and pectin biopolymers with different molecular weight were produced on a pilot-scale from discarded carrots. The pulp was subjected to hydrothermal extraction on a pilot plant operating in cycles of three flow-through reactors in series at 140 and 180 °C. Two operational modes of start-up (with and without water pre-filling) were tested, showing that it influences the final hydrolysate concentration and the stability of the system. Free sugars, arabinogalactan hemicellulose and homogalacturonan pectin were produced at maximum recoveries of 379.5 g/kg dry pulp, 81.0 g/kg dry pulp and 5.35 g/kg dry pulp, respectively, with the extraction of arabinogalactan reaching a yield of 96.1 % (w/w). The extracted biopolymers were separated and purified through ultrafiltration and diafiltration cycles using a multi-membrane system (30, 10, 1 kDa). Ultrafiltration and especially diafiltration allowed going from extracts with molecular weight, polydispersity and purity values of 14.77 kDa, 19.2 and 22.2 % w/w (140 °C extract) and 8.08 kDa, 18.2 and 14.9 % w/w (180 °C extract), to fractions with values of 80.36 kDa, 67.77 kDa, 9.85 kDa, 5.23 kDa and 3.86 kDa (molecular weight), 1.3 – 3.8 (polydispersity), and 64.5 – 100 % w/w (purity). The five fractions were freeze-dried and spray-dried. The pilot-scale system allowed the production of purified biopolymer fractions of high purity, different average molecular weight, and in sufficient quantity for their subsequent transformation into products such as biodegradable films.

Improving emulsification properties of alkaline protein extract from green tea residue by enzymatic methods

Alkaline extraction is an important process in the integrated biorefining of leafy biomass to obtain protein, but the resulting alkaline protein extract (APE11alkaline protein extract: APE.) may have poor emulsification properties for food applications. In this study, the components in the APE fractionations obtained by size exclusion chromatography were determined. The emulsification properties of APE were determined using oil/water with a ratio of 7:3. Whey protein and soybean protein isolate were used as controls while enzymes were used to improve APE's emulsification properties. The results showed that the APE could be divided into three fractions with protein content of 83, 56, and 34%. Carbohydrates mainly derived from homogalacturonan pectin were mostly in Fraction 2, while Fraction 3 consisted of peptides, oligosaccharides, and free polyphenols. The APE had similar emulsification capacity and emulsification stability as those of whey protein and soybean isolate. The emulsion made by the APE had a creaming index of 92% with emulsification activity index value of 44 m2 g−1, and these numbers could retain after storing at 25 °C for 15 days. The emulsification properties of the APE can be further improved by carbohydrate degradation. With the use of Viscozyme® L, the emulsification activity index value of treated APE was increased by 60%, and then still retained at 67 m2 g−1 after storing for 15 days. Treated by either pepsin or alkaline protease, the emulsification properties of APE were decreased, suggesting the key role of protein in APE for emulsification.

Structural Characterization of Polysaccharides Isolated from Panax notoginseng Medicinal Residue and Its Protective Effect on Myelosuppression Induced by Cyclophosphamide.

This study aims to establish the isolation and purification method of polysaccharides from medicinal residue of Panax notoginseng (PPN). The structure and protective effect of PPN on myelosuppression mice were investigated. One neutral polysaccharide (NPPN) and five acidic polysaccharides (APPN I, APPN II-A, APPN II-B, APPN III-A, and APPN III-B) were obtained. The results confirmed that NPPN, APPN I and APPN II-A are glycan with 1, 4 main chains. APPN III-A is a glycan. APPN II-B and APPN III-B are homogalacturonan pectin with 1, 4 main chains. This study demonstrated that NPPN played a bone marrow protective role in myelosuppression mice induced by cyclophosphamide. NPPN could relieve cell cycle arrest, reduce the apoptosis rate of marrow cells, and improve granulocyte-macrophage colony-stimulating (GM-CSF), thermoplastic polyolefin (TPO) and erythropoietin (EPO) serum level, which contributes to promoting the proliferation of hematopoietic cells.

Two \u03b2-glucuronosyltransferases involved in the biosynthesis of type II arabinogalactans function in mucilage polysaccharide matrix organization in Arabidopsis thaliana

BackgroundArabinogalactan-proteins (AGPs) are heavily glycosylated with type II arabinogalactan (AG) polysaccharides attached to hydroxyproline residues in their protein backbone. Type II AGs are necessary for plant growth and critically important for the establishment of normal cellular functions. Despite the importance of type II AGs in plant development, our understanding of the underlying role of these glycans/sugar residues in mucilage formation and seed coat epidermal cell development is poorly understood and far from complete. One such sugar residue is the glucuronic acid residues of AGPs that are transferred onto AGP glycans by the action of β-glucuronosyltransferase genes/enzymes.ResultsHere, we have characterized two β-glucuronosyltransferase genes, GLCAT14A and GLCAT14C, that are involved in the transfer of β-glucuronic acid (GlcA) to type II AGs. Using a reverse genetics approach, we observed that glcat14a-1 mutants displayed subtle alterations in mucilage pectin homogalacturonan (HG) compared to wild type (WT), while glcat14a-1glcat14c-1 mutants displayed much more severe mucilage phenotypes, including loss of adherent mucilage and significant alterations in cellulose ray formation and seed coat morphology. Monosaccharide composition analysis showed significant alterations in the sugar amounts of glcat14a-1glcat14c-1 mutants relative to WT in the adherent and non-adherent seed mucilage. Also, a reduction in total mucilage content was observed in glcat14a-1glcat14c-1 mutants relative to WT. In addition, glcat14a-1glcat14c-1 mutants showed defects in pectin formation, calcium content and the degree of pectin methyl-esterification (DM) as well as reductions in crystalline cellulose content and seed size.ConclusionsThese results raise important questions regarding cell wall polymer interactions and organization during mucilage formation. We propose that the enzymatic activities of GLCAT14A and GLCAT14C play partially redundant roles and are required for the organization of the mucilage matrix and seed size in Arabidopsis thaliana. This work brings us a step closer towards identifying potential gene targets for engineering plant cell walls for industrial applications.

Subcellular coordination of plant cell wall synthesis.

Organelles of the plant cell cooperate to synthesize and secrete a strong yet flexible polysaccharide-based extracellular matrix: the cell wall. Cell wall composition varies among plant species, across cell types within a plant, within different regions of a single cell wall, and in response to intrinsic or extrinsic signals. This diversity in cell wall makeup is underpinned by common cellular mechanisms for cell wall production. Cellulose synthase complexes function at the plasma membrane and deposit their product into the cell wall. Matrix polysaccharides are synthesized by a multitude of glycosyltransferases in hundreds of mobile Golgi stacks, and an extensive set of vesicle trafficking proteins govern secretion to the cell wall. In this review, we discuss the different subcellular locations at which cell wall synthesis occurs, review the molecular mechanisms that control cell wall biosynthesis, and examine how these are regulated in response to different perturbations to maintain cell wall homeostasis.

Hydrothermal production of high-molecular weight hemicellulose-pectin, free sugars and residual cellulose pulp from discarded carrots

Abstract Discarded carrots account for 30% of the total production ending up in landfills, land, or a small part as cattle food. Valorization of discarded carrot pulp was studied by hydrothermal treatment, fractionating free sugars, hemicellulose and pectin in the liquid phase and residual pulp in the solid phase. Extraction took place in flow-through mode at 140, 160 and 180 °C, achieving recoveries up to 211.0 g/kg dry pulp of free sugars, 29.13 g/kg dry pulp of homogalacturonan pectin, and 70.45 g/kg dry pulp of arabinogalactan hemicellulose. The residual pulp reached a cellulose content of 57.5 wt% while before the treatment it was 10.7 wt%. Most of the free sugars were extracted in the preheating stage in batch, so they could be obtained separately from the biopolymers. The flow-through extraction allowed to obtain hemicellulose and pectin of molecular weights >30 kDa. The evolution of different ranges of molecular weight was studied in detail for a better understanding of the phenomenon of autohydrolysis and the link between hemicellulose and pectin. The ample molecular weight distribution in the hydrolyzate allows for a subsequent fractionation via ultrafiltration membranes, to obtain a high molecular weight fraction for applications such as film-forming (in combination with the residual pulp).

Differential localization of cell wall polymers across generations in the placenta of Marchantia polymorpha.

To further knowledge on cell wall composition in early land plants, we localized cell wall constituents in placental cells of the liverwortMarchantia polymorpha L. using monoclonal antibodies (MAbs) in the transmission electron microscope and histochemical staining. The placenta ofM. polymorphais similar to the majority of bryophytes in thatboth generationscontain transfer cells with extensive wall ingrowths.Although the four major cell wall polymers, i.e., cellulose, pectins, hemicelluloses, and arabinogalactan proteins, are present, there are variations in the richness and specificity across generations. An abundance of homogalacturonan pectins in all placental cell walls is consistent with maintaining cell wall permeability and an acidic apoplastic pH necessary for solute transport.Although similar in ultrastructure, transfer cell walls on the sporophyte side in M. polymorpha are enriched with xyloglucans and diverse AGPs not detected on the gametophyte side of the placenta. Gametophyte wall ingrowths are more uniform in polymer composition. Lastly, extensins and callose are not components of transfer cell walls of M. polymorpha,which deviates from studies on transfer cells in other plants. The difference in polymer localizations in transfer cell walls between generations is consistent with directional movement from gametophyte to sporophyte in this liverwort.